Proteins like monoclonal antibodies (mAbs) have mostly charged and polar amino acids at the surface in an aqueous environment (Barlow, D J and Thornton, J M (1986) Biopolymers 25:1717). Because of molecular interaction with the solution components, the surface residues can undergo multiple chemical and enzymatic modifications, leading to a heterogeneous mixture of protein variants with slight differences on their electrostatic surface (Dick, L W et al., (2009) J. Chromatogr. B 877:3841; Liu, H W et al., (2008) Rapid Commun. Mass Spectrom. 22:4081; Miller, A K, et al., (2011) J. Pharm. Sci. 100:2543; Wang, W R et al., (2011) Mol. Immunol. 48:860). Cation-exchange chromatography (CEC) is considered to be the gold standard to profile the charge heterogeneity of protein therapeutics according to a recent review by Vlasak, J and Ionescu, R (2008 Curr. Pharm. Biotechnol. 9:468). The charge sensitive separation method is typically required by the regulatory agencies to ensure the production consistency during manufacturing and to monitor the degradation level of protein therapeutics (Miller, A K, et al., (2011) J. Pharm. Sci. 100:2543; He, X P Z (2009) Electrophoresis 30:714; Sosic, Z et al., (2008) Electrophoresis 29:4368; Kim, J et al., (2010) J. Chromatogr. B 878:1973: Teshima, G et al., (2010) J. Chromatogr. A 1218:2091).
Analytical ion exchange chromatography (IEC) methods using a pH gradient have emerged as alternative techniques to conventional salt gradient IEC for profiling the charge heterogeneity of therapeutic proteins (Farnan, D and Moreno, G T (2009) Anal. Chem. 81:8846; Tsonev, L I and Hirsh, A G (2008) J. Chromatogr. A 1200:166; Nordborg, A et al., (2009) J. Sep. Sci. 32:2668; Rozhkova, A (2009) J. Chromatogr. A 1216:5989; Rea, J C et al. (2010) J. Pharm. Biomed. Anal. 54:317). In this technique, proteins that are typically loaded on a cation-exchange stationary phase are eluted by increasing the pH of the mobile phase. It has recently been demonstrated that a pH gradient IEC (pH-IEC) method with a relatively broad pH window from 6.0 to 9.5 not only provided better resolution than traditional salt-gradient IEC, but also offered multi-product capability through the analysis of 12 monoclonal antibodies (mAbs) with pI from 7.3 to 9.0 (Farnan, D and Moreno, G T (2009) Anal. Chem. 81:8846). That pH-IEC method is also highly tolerant to sample matrix with varied ionic strengths (0 to 250 mM NaCl) and pH values (5.0 to 8.5) (Farnan, D and Moreno, G T (2009) Anal. Chem. 81:8846). Furthermore, the reported pH-IEC method is not evidently impacted by the column length and chemistry and fast separation with a shorter column can be achieved to improve the throughput of protein variant analysis. According to a recent validation report (Rea, J C et al. (2010) J. Pharm. Biomed. Anal. 54:317), the developed pH-IEC method has shown excellent precision at different chromatography conditions and good linearity at different column loads. Thus the reported pH-IEC method is suitable for routine testing in the biotechnology industry.
Despite the many advantages, the reported pH-IEC method was intended primarily for the mAbs with pI values in the studied range of 7.3 to 9.0. The fact that the elution profile of a mAb can vary with different buffer compositions and concentration, and the pH values at which the mAbs elutes indicates that pH-gradient IEC involves a combined ionic-strength and pH-gradient elution mechanism (Farnan, D and Moreno, G T (2009) Anal. Chem. 81:8846). This is also consistent with Anderson and coworkers' report on pH-gradient anion-exchange chromatography (pH-AIEC) (Anderson, D J and Shan, L (2001) Clin. Chem. 47:128; Shan, L and Anderson, D J (2001) J. Chromatogr. A 909:191; Shan, L and Anderson, D J (2002) Anal. Chem. 74:5641). With an increasing number of mAbs in the development phase in the biotechnology industry, especially more low-pI mAbs that show potentially longer half-life based on the animal studies (Igawa, T. (2010) Protein Eng. Des. Sel. 23:385) there is a need to expand the applicability of pH-IEC methods to a broader range of therapeutic mAbs.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.